Prion diseases are inevitably fatal neurodegenerative zoonotic disorders of animals, including humans, with no known treatment or cure. Prions are comprised largely, if not entirely, of PrPSc, a misfolded form of the normal non-infectious prion protein PrPC. Prion strains are operationally defined by differences in neuropathology that breed true under controlled conditions that are encoded by strain-specific conformations of PrPSc. The long- term goal of this work is to understand the dynamics of prion strains. The objective of this application is to determine if prion strains exist as a mixture of substrains or as a homogeneous population. We hypothesize that prions strains are a dynamic mixture of prion substrains (i.e. quasispecies). This would fundamentally impact our understanding of the mechanism of interspecies transmission, adaptation of prions in response to anti-prion drugs, the ecology of prion transmission in natural settings and may be relevant to other protein misfolding diseases that share prion strain-like features (e.g. Alzheimer?s). Since the structure of PrPSc is poorly defined and technologies do not exist to measure the structure of an individual protein in a mixture, we will test the hypothesis based on the predicted properties of quasispecies that are experimentally feasible. First we will determine the effect of repeated limiting dilutions on prion fitness. Repeated bottleneck passage of a quasispecies leads to a reduction in fitness (i.e. Muller?s ratchet) since, on average, the fitness of an individual infectious unit is lower compared to the fitness of the overall population. We anticipate that repeated transmission of prions at limiting dilutions in PMCA or in cell culture will result in a reduction in the fitness of the prion agent compared to agent passaged at high titer. Second we will establish if biologically cloned prion strains contain substrains. Quasispecies hypothesis predicts that any given prion strain is comprised of a dynamic population of substrains. We will test this by amplifying PrPSc in vitro or passaging in cells under conditions that favor the selection of a minor substrain. In a second series of experiments, we will select for subpopulations of PrPSc that have common shared biochemical features to seed PMCA reactions or prion susceptible RK13 cells. Serial repeated rounds of selection followed by transmission in animals will determine if biologically cloned prion strains contain additional strains and if biochemical selective pressure results in the emergence of strains with the selected properties. The results of these experiments will directly test the hypothesis that prions are quasispecies.